Characterization of Cellulolytic and Xylanolytic Enzymes of Bacillus licheniformis JK7 Isolated from the Rumen of a Native Korean Goat.

Department of Agriculture Biotechnology, Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Science, Seoul National University, Seoul 151-742, Korea.

Abstract

A facultative bacterium producing cellulolytic and hemicellulolytic enzymes was isolated from the rumen of a native Korean goat. The bacterium was identified as a Bacillus licheniformis on the basis of biochemical and morphological characteristics and 16S rDNA sequences, and has been designated Bacillus licheniformis JK7. Endoglucanase activities were higher than those of β-glucosidase and xylanase at all temperatures. Xylanase had the lowest activity among the three enzymes examined. The optimum temperature for the enzymes of Bacillus licheniformis JK7 was 70°C for endoglucanase (0.75 U/ml) and 50°C for β-glucosidase and xylanase (0.63 U/ml, 0.44 U/ml, respectively). All three enzymes were stable at a temperature range of 20 to 50°C. At 50°C, endoglucanse, β-glucosidase, and xylanase had 90.29, 94.80, and 88.69% residual activity, respectively. The optimal pH for the three enzymes was 5.0, at which their activity was 1.46, 1.10, and 1.08 U/ml, respectively. The activity of all three enzymes was stable in the pH range of 3.0 to 6.0. Endoglucanase activity was increased 113% by K(+), while K(+), Zn(+), and tween 20 enhanced β-glucosidase activity. Xylanase showed considerable activity even in presence of selected chemical additives, with the exception of Mn(2+) and Cu(2+). The broad range of optimum temperatures (20 to 40°C) and the stability under acidic pH (4 to 6) suggest that the cellulolytic enzymes of Bacillus licheniformis JK7 may be good candidates for use in the biofuel industry.

Bacterial growth curve ( ) and endoglucanase activity ( ) of Bacillus licheniformis JK7. The cell growth was determined by measuring the OD600 of the cell culture. Enzyme activity was determined using the culture supernatants. All experiments were performed in triplicate. The data points and error bars indicate the average values and standard errors.

Temperature and pH effects on endoglucanase, β-glucosidase and xylanase of Bacillus licheniformis JK7 activity (A and C) their stability (B and D). Enzyme activity of the culture supernatants was determined at 24 h. All assays were performed in triplicate. The data points and error bars indicate the average values and standard errors. a,b,c,d,e,f,g Indicates a significantly (p<0.05) different activity influenced by temperature or pH in the same enzyme group. x,y,z Indicates a significantly (p<0.05) different activity between different enzymes within same temperature or pH. 1 NS means not significant.

Temperature and pH effects on endoglucanase, β-glucosidase and xylanase of Bacillus licheniformis JK7 activity (A and C) their stability (B and D). Enzyme activity of the culture supernatants was determined at 24 h. All assays were performed in triplicate. The data points and error bars indicate the average values and standard errors. a,b,c,d,e,f,g Indicates a significantly (p<0.05) different activity influenced by temperature or pH in the same enzyme group. x,y,z Indicates a significantly (p<0.05) different activity between different enzymes within same temperature or pH. 1 NS means not significant.

Effect of chemical additives on endoglucanase, β-glucosidase, xylanase activity of Bacillus licheniformis JK7. Residual activity was calculated as relative (%) considering control as 100%. All assays were performed in triplicate. The data points and error bars indicate the average values and standard errors. a,b,c,d,e,f Indicates a significantly (p<0.05) different enzyme activity compared to control.